Synthesis and Topoisomerase I inhibitory properties of klavuzon derivatives

Klavuzon is a naphthalen-1-yl substituted α,β-unsaturated δ-lactone derivative, and is one of the anti-proliferative members of this class of compounds. Asymmetric and racemic syntheses of novel α,β-unsaturated δ-lactone derivatives are important to investigate their potential for the treatment of cancer. In this study, asymmetric and racemic syntheses of heteroatom-substituted klavuzon derivatives are reported. The syntheses were completed by a well-known three-step procedure. Anti-proliferative activity of seven novel racemic klavuzon derivatives were reported against MCF-7, PC3, HCT116 p53+/+ and HCT116 p53−/− cancer cell lines. Topoisomerase I inhibitory properties of 5,6-dihydro-2H-pyran-2-one derivatives were also studied.     

A novel and one-pot synthesis of new 1-tosyl pyrrol-2-one derivatives and analysis of carbonic anhydrase inhibitory potencies

Here we propose a novel one-pot synthesis of new tosyl-pyrrole derivatives. By means of the new developed method, pyrrole derivatives were synthesized at room temperature in a single step, and a useful method is proposed for the synthesis of similar compounds. Moreover, inhibitions of two human cytosolic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I and II by 1-tosyl-pyrrole and 1-tosyl-pyrrol-2-on derivatives were investigated. 1-Tosyl-pyrrole, 1-tosyl-1H-pyrrol-2(5H)-one, 5-hydroxy-1-tosyl-1H-pyrrol-2(5H)-one and 5-oxo-1-tosyl-2,5-dihydro-1H-pyrrol-2-yl acetate showed inhibitory action with K_i values in the range of 14.6–42.4 μM for hCA I and 0.53–37.5 μM for hCA II, respectively. All pyrrole derivatives were competitive inhibitors with 4-nitrophenylacetate as substrate. Some new synthesized pyrrole derivatives showed very effective hCA II inhibitory effects, in the same range as the clinically used sulfonamide acetazolamide, and might be used as leads for generating enzyme inhibitors targeting other CA isoforms.     

Synthesis and anticancer activity of bis-benzo[d][1,3]dioxol-5-yl thiourea derivatives with molecular docking study

New thiourea derivatives incorporating two benzo[d][1,3]dioxol-5-yl moieties have been synthesized through the reaction of two molecules of benzo[d][1,3]dioxol-5-yl isothiocyanate with one molecule of various diamino derivatives. The synthesized compounds were examined for their cytotoxic effects using SRB assay on three cancer cell lines HepG2, HCT116 and MCF-7. Most of compounds showed significant antitumor activity and some compounds showed strong results greater than the reference drug. As example, IC₅₀ values of 1,1′-(1,4-phenylene)bis(3-(benzo[d][1,3]dioxol-5-yl)thiourea) 5 were 2.38 µM for HepG2, 1.54 µM for HCT116 and 4.52 µM for MCF7, while the IC₅₀ values of standard drug doxorubicin were 7.46, 8.29 and 4.56 µM, respectively. Interestingly, these compounds were non cytotoxic toward the tested normal cell line (IC₅₀ value > 150 µM). The anticancer mechanisms were studied via EGFR inhibition assessment, annexin V-FITC apoptosis assessment, cell cycle analysis and study the effect on mitochondrial apoptosis pathway proteins Bax and Bcl-2 as well as molecular docking studies.     

Simvastatin induced HCT116 colorectal cancer cell apoptosis through p38MAPK-p53-survivin signaling cascade

Background

Statins, the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors with cholesterol-lowering properties, were recently shown to exhibit anti-cancer effects. However, the molecular mechanism underlying statin-induced cancer cell death remains to be elucidated. Elevated level of survivin is often found over-expressed in human cancers and has been implicated in the progression of tumorigenesis. Given its central role in cell division and action as an apoptosis suppressor, survivin represents a potential molecular target in cancer management.

Methods

In this study, we explored the underlying mechanisms in simvastatin-induced HCT116 colorectal cancer cell apoptosis.

Results

Simvastatin decreased cell viability and induced cell apoptosis in HCT116 cells. These results are associated with the modulation of p21^cip/Waf1 and survivin. Survivin knockdown using survivin siRNAs also decreased cell viability and induced cell apoptosis. Simvastatin's actions on p21^cip/Waf1, survivin and apoptosis were reduced in p53 null HCT116 cells. Simvastatin caused an increase in p53 phosphorylation and acetylation. In addition, simvastatin activated p38 mitogen-activated protein kinase (p38MAPK), whereas an inhibitor of p38MAPK signaling abrogated simvastatin's effects of increasing p53 and p21^cip/Waf1 promoter luciferase activity. Cell viability and survivin promoter luciferase activity in the presence of simvastatin were also restored by p38MAPK inhibitor. Furthermore, Sp1 binding to the survivin promoter region decreased while p53 and p63 binding to the promoter region increased after simvastatin exposure.

Conclusions

Simvastatin activates the p38MAPK-p53-survivin cascade to cause HCT116 colorectal cancer cell apoptosis.

General significance

This study delineates, in part, the underlying mechanisms of simvastatin in decreasing survivin and subsequent colorectal cancer cell apoptosis.     

Synthesis and biological evaluation of hesperetin derivatives as agents inducing apoptosis

A flavanone, hesperetin, has been known to exert antitumor activity by inducing apoptosis. To find hesperetin derivatives showing better antitumor activity, 12 derivatives were designed and synthesized. Their antitumor activities were measured using a long-term survival clonogenic assay. Among the compounds, K-5b, hesperetin-7-butyrate, showed the half-maximal cell growth inhibitory concentration three times as low as that of hesperetin. To compare the cytotoxicity of hesperetin and K-5b, the HCT116 human colon cancer cell line was treated with various concentrations of each compound. K-5b decreased the cell viability to a larger extent than hesperetin and triggered apoptosis more efficiently than hesperetin in an apoptosis detection assay using fluorescein isothiocyanate-labeled annexin V. Immunoblotting analysis showed that K-5b promoted caspase-mediated apoptosis more efficiently than hesperetin. Because hesperetin has been reported to induce apoptosis through the activation of the c-Jun N-terminal kinase (JNK) pathway, we tested whether K-5b activates JNKs. K-5b stimulated JNK1 and JNK2 more efficiently than hesperetin as shown by western blot analysis. In conclusion, hesperetin derivatives exerting better antitumor activity than hesperetin by inducing apoptosis were found.     

Synthesis and anti-proliferative activity of a small library of 7-substituted 5H-pyrrole [1,2-a][3,1]benzoxazin-5-one derivatives

In this study, we investigate the anti-proliferative activity of a small library of 7-substituted 5H-pyrrolo[1,2-a][3,1]benzoxazin-5-one derivatives, against a panel of human cancer cell lines. We reported the synthesis of these compounds in a previous work. 7-Bromo-5H-benzo[d]pyrrolo[2,1-b][1,3]oxazin-5-one showed a promising anti-proliferative effect. As starting material for Suzuki-Miyaura cross coupling reaction, it was selected for the design and the synthesis of six further derivatives, with the aim to better define structure-activity relationships. The anti-proliferative MTT assay revealed a dose-dependent reduction of cell viability, especially for 7-([1,1′-biphenyl]-4-yl)-5H-benzo[d]pyrrolo[2,1-b][1,3]oxazin-5-one. Cell cycle and western blotting analysis suggested apoptosis as possible mechanism for its anti-proliferative activity. These preliminary results encourage our interest for further optimizations.     

Design,synthesis and antitumour and anti-angiogenesis evaluation of 22 moscatilin derivatives

Two series of moscatilin derivatives were designed, synthesized and evaluated as anti-tumor and anti-angiogenesis agents. Most of these compounds showed moderate-to-obvious cytotoxicity against five cancer cell lines (A549, HepG2, MDA-MB-231, MKN-45, HCT116). Among these cell lines, compounds had obvious effects on HCT116. Especially for 8Ae, the IC₅₀ was low to 0.25 μM. 8Ae can inhibit the viability and induce the apoptosis of HCT116 cells but exhibit no cytotoxic activity in noncancerous NCM460 colon cells. 8Ae can also arrest the G2/M cell cycle in HCT116 cells by inhibiting the α-tubulin expression. Zebrafish bioassay-guided screen showed the 22 moscatilin derivatives had potent anti-angiogenic activities and compound 8Ae had better activities than positive compound. Molecular docking indicated 8Ae interacted with tubulin at the affinity of −7.2 Kcal/mol. In conclusion, compound 8Ae was a potential antitumor and anti-angiogenesis candidate for further development.     

Synthesis and evaluation of modified chalcone based p53 stabilizing agents

Tumor suppressor protein p53 induces cell cycle arrest and apoptotic cell death in response to various cellular stresses thereby preventing cancer development. Activation and stabilization of p53 through small organic molecules is, therefore, an attractive approach for the treatment of cancers retaining wild-type p53. In this context, a series of nineteen chalcones with various substitution patterns of functional groups including chloro, fluoro, methoxy, nitro, benzyloxy, 4-methyl benzyloxy was prepared using Claisen-Schmidt condensation. The compounds were characterized using NMR, HRMS, IR and melting points. Evaluation of synthesized compounds against human colorectal (HCT116) and breast (CAL-51) cancer cell lines revealed potent antiproliferative activities. Nine compounds displayed GI₅₀ values in the low micromolar to submicromolar range; for example (E)-1-phenyl-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (SSE14108) showed GI₅₀ of 0.473 ± 0.043 µM against HCT116 cells. Further analysis of these compounds revealed that (E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (SSE14105) and (E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (SSE14106) caused rapid (4 and 8-h post-treatment) accumulation of p53 in HCT116 cells similar to its induction by positive control, Nutlin-3. Such activities were absent in 3-(4-methoxyphenyl)propiophenone (SSE14106H2) demonstrating the importance of conjugated ketone for antiproliferative and p53 stabilizing activity of the chalcones. We further evaluated p53 levels in the presence of cycloheximide (CHX) and the results showed that the p53 stabilization was regulated at post-translational level through blockage of its degradation. These chalcones can, therefore, act as fragment leads for further structure optimization to obtain more potent p53 stabilizing agents with enhanced anti-proliferative activities.     

Discovery of N-aryl-9-oxo-9H-fluorene-1-carboxamides as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 1. Structure–activity relationships of the carboxamide group

N-(2-Methylphenyl)-9-oxo-9H-fluorene-1-carboxamide (2a) was identified as a novel apoptosis inducer through our caspase- and cell-based high-throughput screening assay. Compound 2a was found to be active with sub-micromolar potencies for both caspase induction and growth inhibition in T47D human breast cancer, HCT116 human colon cancer, and SNU398 hepatocellular carcinoma cancer cells. It arrested HCT116 cells in G₂/M followed by apoptosis as assayed by the flow cytometry. Structure–activity relationship (SAR) studies of the carboxamide group identified the lead compound N-(2-(1H-pyrazol-1-yl)phenyl)-9-oxo-9H-fluorene-1-carboxamide (6s). Compound 6s, with increased aqueous solubility, was found to retain the broad activity in the caspase activation assay and in the cell growth inhibition assay with sub-micromolar EC₅₀ and GI₅₀ values in T47D, HCT116, and SNU398 cells, respectively.     

The relative contribution of pro-apoptotic p53 target genes in the triggering of apoptosis following DNA damage in vitro and in vivo

The p53 pathway displays a large degree of redundancy in the expression of a number of pro-apoptotic mechanisms following DNA damage that, among others, involves increased expression of several pro-apoptotic genes through transactivation. Spatial and temporal cellular contexts contribute to the complexity of the regulation of apoptosis, hence different genes may show a cell- and tissue-dependent specificity with regard to the regulation of cell death and act in concert or show redundancy with one and another. We used siRNA technology to assess the effect of multiple ablations of documented pro-apoptotic p53 target genes (PPG) in the colorectal cancer cell line HCT116 and generated mice deficient in both of the extrinsic and intrinsic PPGs genes Dr5 and Puma following treatment with chemotherapeutics and ionizing radiation. DR5, Fas, Bax, Bad, Puma and Bnip3L were induced by 5-FU and adriamycin (ADR) in HCT116 cells in a p53-dependent manner. The resulting caspase 3/7 activity in HCT116 cells following treatment were suppressed by ablated expression of the PPGs in the extrinsic as well as the intrinsic pathway. To our surprise, knocking-down any of the PPGs concomitantly with DR5 did not further inhibit caspase 3/7 activity whereas inhibiting DR5-expression in HCT116Bax knockdown (kd) and HCT116Fas kd did, suggesting that these genes act downstream or in synergy with DR5. This was supported by our in vivo observations, since Puma and Dr5 were equally efficient in protecting cells of the spleen from sub-lethal radiation-induced apoptosis but less effective compared with irradiated p53^−/− mice. To our surprise, Dr5^−/−; Puma^−/− mice did not show additive protection from radiation-induced apoptosis in any of the investigated organs. Our data indicates that the intrinsic pathway may rely on extrinsic signals to promote cell death in a cell- and tissue-dependent manner following DNA damage. Furthermore, p53 must rely on mechanisms independent of DR5 and PUMA to initiate apoptosis following γ-radiation in the spleen and thymus in vivo.Key words: p53, KILLER/DR5, PUMA, apoptosis, DNA damage     

Inhibitory effect of novel somatostatin peptide analogues on human cancer cell growth based on the selective inhibition of DNA polymerase β

The present study was designed to investigate the anticancer activity of novel nine small peptides (compounds 1–9) derived from TT-232, a somatostatin structural analogue, by analyzing the inhibition of mammalian DNA polymerase (pol) and human cancer cell growth. Among the compounds tested, compounds 3 [tert-butyloxycarbonyl (Boc)-Tyr-Phe-1-naphthylamide], 4 (Boc-Tyr-Ile-1-naphthylamide), 5 (Boc-Tyr-Leu-1-naphthylamide) and 6 (Boc-Tyr-Val-1-naphthylamide) containing tyrosine (Tyr) but no carboxyl groups, selectively inhibited the activity of rat pol β, which is a DNA repair-related pol. Compounds 3–6 strongly inhibited the growth of human colon carcinoma HCT116 p53^+/+ cells. The influence of compounds 1–9 on HCT116 p53^?/? cell growth was similar to that observed for HCT116 p53^+/+ cells. These results suggest that the cancer cell growth suppression induced by these compounds might be related to their inhibition of pol. Compound 4 was the strongest inhibitor of pol β and cancer cell growth among the nine compounds tested. This compound specifically inhibited rat pol β activity, but had no effect on the other 10 mammalian pols investigated. Compound 4 combined with methyl methane sulfonate (MMS) treatment synergistically suppressed HCT116 p53^?/? cell growth compared with MMS alone. This compound also induced apoptosis in HCT116 cells with or without p53. From these results, the influence of compound 4, a specific pol β inhibitor, on the relationship between DNA repair and cancer cell growth is discussed.     

Polo-like kinase 1 inhibitors,mitotic stress and the tumor suppressor p53

Polo-like kinase 1 has been established as one of the most attractive targets for molecular cancer therapy. In fact, multiple small-molecule inhibitors targeting this kinase have been developed and intensively investigated. Recently, it has been reported that the cytotoxicity induced by Plk1 inhibition is elevated in cancer cells with inactive p53, leading to the hypothesis that inactive p53 is a predictive marker for the response of Plk1 inhibition. In our previous study based on different cancer cell lines, we showed that cancer cells with wild type p53 were more sensitive to Plk1 inhibition by inducing more apoptosis, compared with cancer cells depleted of p53. In the present work, we further demonstrate that in the presence of mitotic stress induced by different agents, Plk1 inhibitors strongly induced apoptosis in HCT116 p53^+/+ cells, whereas HCT116 p53^−/− cells arrested in mitosis with less apoptosis. Depletion of p53 in HCT116 p53^+/+ or U2OS cells reduced the induction of apoptosis. Moreover, the surviving HCT116 p53^−/− cells showed DNA damage and a strong capability of colony formation. Plk1 inhibition in combination with other anti-mitotic agents inhibited proliferation of tumor cells more strongly than Plk1 inhibition alone. Taken together, the data underscore that functional p53 strengthens the efficacy of Plk1 inhibition alone or in combination by strongly activating cell death signaling pathways. Further studies are required to investigate if the long-term outcomes of losing p53, such as low differential grade of tumor cells or defective DNA damage checkpoint, are responsible for the cytotoxicity of Plk1 inhibition.     

The p53-p21WAF1 checkpoint pathway plays a protective role in preventing DNA rereplication induced by abrogation of FOXF1 function

We previously identified FOXF1 as a potential tumor suppressor gene with an essential role in preventing DNA rereplication to maintain genomic stability, which is frequently inactivated in breast cancer through the epigenetic mechanism. Here we further addressed the role of the p53-p21^WAF1 checkpoint pathway in DNA rereplication induced by silencing of FOXF1. Knockdown of FOXF1 by small interference RNA (siRNA) rendered colorectal p53-null and p21^WAF1-null HCT116 cancer cells more susceptible to rereplication and apoptosis than the wild-type parental cells. In parental HCT116 cells with a functional p53 checkpoint, the p53-p21^WAF1 checkpoint pathway was activated upon FOXF1 knockdown, which was concurrent with suppression of the CDK2-Rb cascade and induction of G₁ arrest. In contrast, these events were not observed in FOXF1-depleted HCT116-p53−/− and HCT116-p21−/− cells, indicating that the p53-dependent checkpoint function is vital for inhibiting CDK2 to induce G₁ arrest and protect cells from rereplication. The pharmacologic inhibitor (caffeine) of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases abolished activation of the p53-p21^WAF1 pathway upon FOXF1 knockdown, suggesting that suppression of FOXF1 function triggered the ATM/ATR-mediated DNA damage response. Cosilencing of p53 by siRNA synergistically enhanced the effect of FOXF1 depletion on the stimulation of DNA rereplication and apoptosis in wild-type HCT116. Finally, we show that FOXF1 expression is predominantly silenced in breast and colorectal cancer cell lines with inactive p53. Our study demonstrated that the p53-p21^WAF1 checkpoint pathway is an intrinsically protective mechanism to prevent DNA rereplication induced by silencing of FOXF1.     

Synthesis,biological evaluation,and in silico studies of new CDK2 inhibitors based on pyrazolo[3,4-d]pyrimidine and pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine scaffold with apoptotic activity

Cyclin-dependent kinase inhibition is considered a promising target for cancer treatment for its crucial role in cell cycle regulation. Pyrazolo pyrimidine derivatives were well established for their antitumor activity via CDK2 inhibition. In this research, new series of pyrazolopyrimidine derivatives (4–15) was designed and synthesised as novel CDK2 inhibitors. The anti-proliferative activities against MCF-7, HCT-116, and HepG-2 were used to evaluate their anticancer activity as novel CDK2 inhibitors. Most of the compounds showed superior cytotoxic activity against MCF-7 and HCT-116 compared to Sorafenib. Only compounds 8, 14, and 15 showed potent activity against HepG-2. The CDK2/cyclin A2 enzyme inhibitory activity was tested for all synthesised compounds. Compound 15 showed the most significant inhibitory activity with IC₅₀ 0.061 ± 0.003 µM. It exerted remarkable alteration in Pre G1 and S phase cell cycle progression and caused apoptosis in HCT cells. In addition, the normal cell line cytotoxicity for compound 15 was assigned revealing low cytotoxic results in normal cells rather than cancer cells. Molecular docking was achieved on the designed compounds and confirmed the two essential hydrogen binding with Leu83 in CDK2 active site. In silico ADMET studies and drug-likeness showed proper pharmacokinetic properties which helped in structure requirements prediction for the observed antitumor activity.     

Three new compounds from Artemisia anomala and their antitumor activities

Three new compounds were isolated from Artemisia anomala, and their structures were determined using HR-ESI-MS, IR, UV, and NMR. The antitumor activities of the three compounds were evaluated in the human lung cancer cell line A549 and the human colorectal cancer cell line HCT116. The results showed that compound 2 significantly inhibited cell viability and proliferation and promoted apoptosis of HCT116 and A549 cells, suggesting that compound 2 may be used for colon and lung cancer treatments in clinical practice.     

Delivery of sTRAIL variants by MSCs in combination with cytotoxic drug treatment leads to p53-independent enhanced antitumor effects

Mesenchymal stem cells (MSCs) are able to infiltrate tumor tissues and thereby effectively deliver gene therapeutic payloads. Here, we engineered murine MSCs (mMSCs) to express a secreted form of the TNF-related apoptosis-inducing ligand (TRAIL), which is a potent inducer of apoptosis in tumor cells, and tested these MSCs, termed MSC.sTRAIL, in combination with conventional chemotherapeutic drug treatment in colon cancer models. When we pretreated human colorectal cancer HCT116 cells with low doses of 5-fluorouracil (5-FU) and added MSC.sTRAIL, we found significantly increased apoptosis as compared with single-agent treatment. Moreover, HCT116 xenografts, which were cotreated with 5-FU and systemically delivered MSC.sTRAIL, went into remission. Noteworthy, this effect was protein 53 (p53) independent and was mediated by TRAIL-receptor 2 (TRAIL-R2) upregulation, demonstrating the applicability of this approach in p53-defective tumors. Consequently, when we generated MSCs that secreted TRAIL-R2-specific variants of soluble TRAIL (sTRAIL), we found that such engineered MSCs, labeled MSC.sTRAIL^DR5, had enhanced antitumor activity in combination with 5-FU when compared with MSC.sTRAIL. In contrast, TRAIL-resistant pancreatic carcinoma PancTu1 cells responded better to MSC.sTRAIL^DR4 when the antiapoptotic protein XIAP (X-linked inhibitor of apoptosis protein) was silenced concomitantly. Taken together, our results demonstrate that TRAIL-receptor selective variants can potentially enhance the therapeutic efficacy of MSC-delivered TRAIL as part of individualized and tumor-specific combination treatments.